Multiple Rab GTPase binding sites in GCC185 suggest a model for vesicle tethering at the trans-Golgi.
GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.
Hayes
Garret L
GL
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
Brown
Frank C
FC
Haas
Alexander K
AK
Nottingham
Ryan M
RM
Barr
Francis A
FA
Pfeffer
Suzanne R
SR
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism* / Q000648:ultrastructure
C472299:GCC2 protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; C097313:ADP-ribosylation factor related proteins; D020727:ADP-Ribosylation Factors; D020691:rab GTP-Binding Proteins